The emergence of in vivo resistance of Plasmodium falciparum malaria to the combination drug pyrimethamine-sulfadoxine (PS) is a major public health threat in Africa. The overall aim of this project is to define the mechanisms(s) of this resistance through a combination of field studies in Africa and laboratory studies at the University of Maryland. The first specific aim is to test the hypothesis that point mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), the target enzymes of pyrimethamine and sulfadoxine, respectively, are associated with in vivo resistance to PS. In prospective studies in Africa, the in vivo response to PS of individual P. falciparum infections will be correlated with the presence or absence o specific mutations in DHFR and DHPS. Simple and reliable molecular assays for epidemiologic mapping of PS-resistant malaria will be developed, and the predictive value of these assays for in vivo PS resistance and therapeutic failure established. The proposed research will also study the mechanisms by which drug pressure causes the spread of resistance-conferring mutations both in individual infections and in epidemiologic populations. The second aim is to assess the contribution of other factors to PS resistance, including partial immunity to malaria infection; antagonism of PS by folate or para-aminobenzoic acid (PABA); and differences among individuals in drug bioavailability and metabolism. The role of these factors will be examined through analyses of clinical specimens and parasites from cases o clinically PS-sensitive and PS-resistant malaria. The third aim of the project is to conduct surveillance for new mutations in DHFR and DHPS and assess the role of newly discovered DHFR and DHPS mutations on antifolate resistance through in vitro transfection studies. The proposed research will result in increased understanding of the basic genetic and molecular mechanisms through which drug-resistant malaria develops. These studies are expected to provide tools for the surveillance and control of drug-resistant malaria and to yield information that will aid in designing new antifolate drugs or drug combinations that will be less prone to the development of resistance.